Aspergillus Carneus metabolite Averufanin induced cell cycle arrest and apoptotic cell death on cancer cell lines via inducing DNA damage

Cancer is one of the leading causes of death worldwide, accounting for nearly 10 million deaths in 2020. Current treatment methods include hormone therapy, γ-radiation, immunotherapy, and chemotherapy. Although chemotherapy is the most effective treatment, there are major obstacles posed by resistance mechanisms of cancer cells and side-effects of the drugs, thus the search for novel anti-cancer compounds, especially from natural sources, is crucial for cancer pharmaceutics research. One natural source worthy of investigation is fungal species. In this study, the cytotoxicity of 5 metabolic compounds isolated from filamentous fungus Aspergillus Carneus. Arugosin C, Averufin, Averufanin, Nidurifin and Versicolorin C were analyzed using NCI-SRB assay on 10 different cell lines of breast cancer, ovarian cancer, glioblastoma and non-tumorigenic cell lines. Averufanin showed highest cytotoxicity with lowest IC50 concentrations especially on breast cancer cells. Therefore, Averufanin was further investigated to enlighten cell death and molecular mechanisms of action involved. Cell cycle analysis showed increase in SubG1 phase suggesting apoptosis induction which was further confirmed by Annexin V and Caspase 3/7 Assays. H2A.X staining revealed accumulation of DNA damage in cells treated with Averufanin and finally western blot analysis validated DNA damage response and downstream effects of Averufanin treatment in various signaling pathways. Consequently, this study shows that Averufanin compound induces cell cycle arrest and cell death via apoptosis through causing DNA damage and can be contemplated and further explored as a new therapeutic strategy in breast cancer.

www.nature.com/scientificreports/ inhibitors such as p21 Cip1 and apoptotic proteins such as Bax, PUMA and Noxa, respectively 10 . Although these are the prevalent roles of p53, studies show that these are not the only pathways p53 is involved. Some examples are p53 and lncRNA interactions 11 , miRNA processing 12 and CyclinA2/CDK2 complex activating. The latter was recently demonstrated by a study that Cyclin A2/CDK2 complex is not just an S phase cell cycle regulator but also a DNA damage response and takes a role in double-strand repair coupling with Ku proteins 13 . The mechanisms activating p53 is also widely studied and among several other proteins in regulatory roles, GSK3β phosphorylates p53 so that it is in its active form 14 . GSK3β is also activated under stress and DNA damage conditions, upon dephosphorylation and is also capable of downregulating Cyclin D1 along with the downstream p53-p21-Cyclin D1 pathway 15 . In the presence of an active GSK3β, Cyclin D1 levels decrease affected by both pathways. Even though treatment options emerged rapidly and promisingly in the last years in breast cancer, patients acquire resistance quickly in most of the cases. Due to the status of its receptors, the susceptibility to alternative therapy methods, such as hormonal therapy, can be altered. For instance, an Estrogen Receptor (ER) positive cell population can be treated with drugs like Tamoxifen, only until the cells gain resistance and bypass receptor related therapies. In addition, there are different types of breast cancer classifications due to their receptor expressions such as Triple Negative Breast Cancer (TNBC) cells, HER2/ER/PR negative or positive cells. On the other hand, breast cancer possesses p53 mutations lower than other types of cancer according to several statistical data 10 . This study investigated the cytotoxic effects of 5 compounds isolated from filamentous fungus Aspergillus Carneus. Aspergillus Carneus is primarily a soil fungus that colonizes mostly in tropical or subtropical terrestrial environments but also found and can grow worldwide 16 . Recent studies also found the species in marine environments [17][18][19] . Anti-cancer effects of fungi metabolites is a broad area of research and different molecules have been tested in different cancer cell lines including lung fibroblast cells 19 , pancreatic cancer cells 20 , liver cancer cells 21 and more. Marine fungi secondary metabolites such as the ones in the current study have been previously reported as potential anti-cancer 22 , anti-microbial or anti-oxidant 23 compounds however, there are a large variety of compounds from different species remains to be identified and their effects to be investigated. Aspergillus spp. renders many metabolites, some of which are tested for their cytotoxicity on some tumorigenic cell lines and their respective IC 50 values are reported and reviewed in many studies [24][25][26][27] . There is a study showing anti-tumor growth effects of compound Aspergiolide A isolated from filamentous fungus Aspergillus Glaucus in mice xenograft model 27 . Bioactive metabolites from the species Aspergillus Carneus has not been extensively studied and reported for their anticancer activity and more importantly, other than its isolation and classification there is no publication to date on the anti-cancer, anti-microbial or anti-oxidant activity of Averufanin which makes this study novel in the area.
In this study, 5 chemical compounds were isolated from Aspergillus Carneus and tested against human cancer cell lines. We documented that Averufanin was highly cytotoxic towards breast cancer cells and relatively low toxicity on non-tumorigenic cell line. Treatment with Averufanin resulted in DNA damage, cell cycle arrest and apoptotic cell death on breast cancer cell lines by modulating p53 mediated cell signaling pathways. Therefore, Averufanin can be a novel candidate compound for therapeutic strategies in cancer research, especially holding a potential for breast cancer.

Results
Chemistry. All chemicals were isolated from Aspergillus Carneus species, and their chemical formulas were drawn using ChemDraw is shown in Fig. 1A.
Biology. Cytotoxicity evaluation of compounds on cancer cell lines. Cytotoxic activities of purified compounds obtained from Aspergillus Carneus (Fig. 1A) were initially investigated on breast cancer (MCF7, MDA-MB-213), ovarian cancer (OVCAR3, OVSAHO, KURAMOCHI) cell lines and a non-tumorigenic cell line from gynecological (HGRC1) origin using NCI SRB assay.
From all 5 compounds isolated from Aspergillus Carneus, Averufanin showed significant levels of cytotoxicity whereas other molecules had no notable effects on cell growth (Fig. 1B-G and Table 1). These results indicated Averufanin as the promising compound for targeting cancer cell lines more effectively than the rest of the compounds and breast cancer cells were more sensitive to treatment with Averufanin therefore in the next panel of NCI-SRB assays, two additional breast cancer (T47D, SK-BR-3) and a Glioblastoma cell line U-87 were included as well as non-tumorigenic breast epithelial (MCF12A) cell line. Figure 2 shows that all three cancer cell lines (U-87, T47D, SK-BR-3) were significantly more sensitive to Averufanin treatment than non-tumorigenic MCF12A cell line.
Cell cycle analysis with PI staining and flow cytometry. To further elucidate the effect of Averufanin on cell cycle, breast cancer cells were treated with Averufanin (IC 50 or IC 75 ) or DMSO negative controls for 48 h and stained with a DNA dye, propidium iodide (PI). Propidium iodide, which is a DNA-intercalating fluorescent dye, is frequently used for cell cycle analysis. Cells that are in G1 phase of cell cycle or at quiescent state (G 0 ) contain one DNA copy (2N), whereas cells at G2/M phase have double copies of DNA (4N). Moreover, if the cells are in S phase of the cell cycle, they have varying copy number. Healthy cancer cells would have about 60-70% of the cells to be in G1 phase, 20% in S phase. Moreover, G2/m phase cells would comprise 20% of the total cell population. During apoptosis, one of the cellular responses is DNA fragmentation into smaller pieces, which is also visible in a PI Flow Cytometry assay as the "SubG0/G1" phase. Experiment results from this assay showed significant increase in SubG1 phase in cells treated with Averufanin compared to DMSO negative controls on both cell lines (Fig. 3A) which also suggested apoptotic cell death induction in those cells which was further analyzed with Annexin V and caspase activities in the following experiments. To further show cell death and cell morphology changes induced, brightfield microscopy images of cells under treatment were included. (Fig. 3B)

Characterization of cell death induced by Averufanin.
To determine the cell death mechanism induced, MUSE Cell Analyzer was used. Human breast cancer cells were treated with Averufanin at their respective IC 75 values for 48 h and DMSO was used as negative control. Annexin V & Dead Cell analysis shows cells positive for Annexin V, a marker for apoptosis on the outer membrane of the cells. Figure 4 shows early and late apoptotic breast cancer cells when treated with Averufanin. In order to further assess the effect of Averufanin on apoptotic cell death, the activities of apoptosis pathway proteins Caspases 3 and 7 were investigated using MUSE Cell Analyzer Caspase3/7 Assay. Breast cancer cells were treated with Averufanin (IC 75 concentrations, Table 1) or DMSO negative control for 48 h. Cells were later stained for caspase 3 and 7 and analyzed. Figure 5 showed that treatment of (A) T47D, (B) MCF-7, and (C) SK-BR-3 cells with Averufanin resulted in occurrence of more apoptotic cells than with DMSO controls.
Accumulation of DNA damage. Phosphorylation of histone H2AX (γ-H2Ax) is a well-established marker of accumulation of dsDNA breaks. Therefore, human breast cancer cells were treated with Averufanin or DMSO negative control for 48 h and then stained with both DAPI and γ-H2Ax antibody, blue and red respectively. Our results indicated that Averufanin treatment resulted in accumulation of γ-H2Ax positive cells when compared to DMSO negative controls (Fig. 6).

Cellular pathways targeted by Averufanin.
Based on the previous data on apoptotic induction, initially proteins involved in apoptosis pathway such as PARP, or p53 were analyzed. Western blot analysis on breast cancer cells treated with Averufanin reveled increase in phosphorylation of p53 (Fig. 7A) and in cleavage of PARP (Fig. 7B) compared to DMSO controls which supports induction of apoptosis. Furthermore, GSK3β and p-GSK3β was investigated to see if Averufanin treatment affected its phosphorylation. Dephosphorylation of Ser9 residue, which makes GSK3β active was observed. (Fig. 7C). Based on the flow cytometry results indicating cell cycle arrest, effect of the compound on Cyclin D1, which is a cell cycle regulator protein and an active GSK3β target was also investigated, and results showed significant decrease in Cyclin D1 levels upon treatment with Averufanin compared to DMSO controls (Fig. 7D). Another DNA damage response and cell cycle regulatory protein  www.nature.com/scientificreports/ Cyclin A2 and its cyclin dependent kinase CDK2 was analyzed, and their levels showed an apparent increase, consistent with the DNA damage response expected. (Fig. 7E,F).

Discussion
In 2020 Breast cancer was the world's most prevalent cancer and deaths caused by it endures to be at an important rate even with all the treatment options. Therefore, search for novel anti-cancer compounds keeps its importance. One of the natural sources worthy of investigating is fungal species and their metabolites mycotoxins, which are natural protective molecules for fungus itself in their habitats, allowing fungus to toxicant other organisms that may grow in the vicinity and act invasive. This study initially investigated 5 compounds that are metabolites of filamentous fungus Aspergillus Carneus and further focused on the compound Averufanin. Averufanin was not only highly cytotoxic for almost all cancer cell lines in our study but also less toxic for non-tumorigenic cell lines MCF12A, HGRC1. Only cell line that had higher IC 50 concentration than nontumorigenic cell lines in the panel was the triple negative cell line MDA-MB-231. TNBC is the most lethal subtype of breast cancer because it has high heterogeneity and aggressive nature 28 , and lack of treatment options still remains an obstacle to overcome. Although TNBC is considered a histological subtype it also has subtypes    29 . This level of heterogeneity makes it harder to emphasize any one single differential pathway or trait and target TNBCs accordingly 30 . Same reason complicates our discussion for Averufanin's mechanism of effecting cells. Although the lowest IC 50 concentrations come from cell lines positive for one or more receptors among estrogen receptor (ER), progesterone receptor (PR), human epidermal growth receptor-2 (HER-2), and one is tempted to make the deduction of correlating Averufanin with receptor status, this hypothesis needs further investigation and is worth looking into in the future. In addition to this compliant feature as an anti-cancer agent, it is also novel because it had not been studied in any field, including antioxidant, agriculture, cancer, and toxicity research. Our study shows that it is indeed a candidate for novel anti-cancer compounds however extensive studies in other fields as well can be adjuvant and elucidative. To improve the understanding of the underlying mechanisms of how Averufanin functions there can be additional experiments using different cell lines and methods in the future, allowing the compound to gain recognition and widely obtained, studied and used in the appropriate areas.
This study covers some of the pathways Averufanin affects in breast cancer cell line T47D. Obtained by the extensive western blot results, Fig. 7G demonstrates a possible mechanism of action for Averufanin on breast cancer cells represented by T47D. Averufanin induced cellular stress and DNA damage on breast cancer cells shown by γH2Ax staining. It is not uncommon for mycotoxin and their precursors to induce DNA damage, reactive oxygen species (ROS) by causing oxidative stress or alteration of mitochondrial function with unknown ways of entering or triggering cells 31 . This DNA damage resulted in the dephosphorylation of p-GSKβ in Ser9 residue 32 , allowing it to be active. Active GSK3β and possibly other DNA damage response elements 33 increased p53 levels and phosphorylation of the upregulated tumor suppressor in Ser15 residue 34 occurred. Both p-p53 35 and GSK3β 15 downregulated cell cycle regulatory protein Cyclin D1 36 , delaying passing to the S phase of cells and resulted in a cell cycle arrest at SubG1 and G1 phase, supported by the PI staining experiments. Meanwhile p-p53 and possibly other DNA damage response elements also effected DNA damage repair elements 13 (as well as being S phase cell cycle regulatory proteins) Cyclin A2 and CDK2 and their levels elevated. Apoptosis regulator p53 also activated the caspase3/7 cascade 37,38 , as shown by the MUSE analysis, resulting in PARP cleavage and programmed cell death, apoptosis. Consequently, our data suggested that Averufanin can be contemplated and further explored as a new therapeutic strategy as an anti-cancer compound, especially for breast cancer.

Methods
Chemistry. Cultivation, isolation and structure elucidation. Aspergillus Carneus which was isolated from marine sponge Agelas Oroides previously, was cultivated in rice medium (100 g rice and 110 ml distilled water) that was autoclaved for 20 min at 121 °C before cultivation through 4 weeks. Then, medium was extracted with Blue represents DAPI and red represents γ-H2Ax which increases as a DNA damage response (DDR). Images were obtained from the confocal microscopy through LasX and dye intensities were evaluated with ImageJ. H2A.X intensities were normalized to DAPI, graphics and statistical analysis were conducted with GraphPad PRISM. Two-tailed, unpaired t-test was applied between DMSO Control and treatment groups (*p < 0.05).  www.nature.com/scientificreports/ 500 ml ethyl acetate three times and organic solvent was dried under vacuum at 40 °C. The concentrated crude extract was fractioned between n-hexane and 90% aqueous methanol. Methanolic phase dried under vacuum at 40 °C and the metabolite isolation was continued with concentrated methanolic fraction 39 . The MeOH extract from solid rice medium (1.5 g) was loaded to vacuum liquid chromatography over silica gel, eluted with a gradient of n-hexane-EtOAc (100:0 to 0:100) followed by DCM-MeOH (100:0 to 0:100) to yield 5 fractions (Fr1-Fr9). Fr1 (500 mg) was fractioned using semi-preparative RP-HPLC eluted with a gradient of MeOH-H 2 O to yield compounds Versicolorin C (5 mg), Averufanin (7 mg), Aruogosin C (3 mg), Nidurufin (3 mg) and Averufin (10 mg).
The NMR spectrums ( 1 H, 13 C) were recorded on a Bruker ARX 600. HPLC analyses were performed a Dionex ultimate 300 LC system coupled with the photodiode array detector (UVD340S). LCMSMS Semipreparative HLPC separations were carried out via he LaChrom-Merck Hitachi system (pump L7100; UV detector L7400; column Eurospher-100C18, 300 × 8 mm, Knauer, Germany) at a flow rate of 5 mL/min. LC-MS spectrums were obtained by using a Thermo Finnigan LCQ Deca mass spectrometer, and HRESIMS spectrums were carried out with a FTHRMS-Orbitrap (Thermo-Finnigan) mass spectrometer. NCI-60 sulforhodamine B assay for in vitro cytotoxicity screening. Cells were inoculated in 96-well plates (1000-5000 cell/well) and grown for 24 h before being treated with increasing concentrations of the compounds (0.2-100 µM or 0.1-50 µM). After 72 h and 48 h of incubation, cells were fixed with 10% (v/v) trichloroacetic acid for 1 h at 4 °C. Then plates were washed five times with deionized water and left to dry. Next day, cells were stained with 0.4% (m/v) of Sulforhodamine B in 1% acetic acid solution in the dark at room temperature for 30 min. The plates washed five times with 1% acetic acid before air-drying. Bound SRB was dissolved in 10 mM Tris-base solution. The absorbance was read in a plate reader at 515 nm 45 . DMSO was used as a negative control. The experiment was performed in triplicates. . Cell lysates containing 20 µg protein were mixed with 4X Laemmli Sample Buffer, 1/6 β-Mercaptoethanol and denatured for 6 min in 95 °C. Samples were loaded to BIORAD Precast gels of 15 wells (4-15%). After electrophoresis, the proteins were transferred to PVDF membranes (BIORAD) with Semidry Transfer System (BIORAD), followed by overnight incubation in blocking solution (5% Blotting Grade Blocker in 1× TBS-T (0.2% tween)). PARP (Cell signaling), p53 (Cell Signaling), Phospho-p53 (Ser15) (Cell Signaling), GSK3β (Cell Signaling), phosphor-GSK3β (Ser9) (Cell Signaling), CyclinD1 (Cell Signaling), Cyclin A2 (Cell Signaling), CDK2 (Cell Signaling) primary antibodies were used in a ratio of 1:500 to 1:1000 in 5% BSA-TBS-T, incubated for 1 h in room temperature. Secondary antibodies, anti-rabbit (Sigma Aldrich), anti-mouse (abcam), were used in 1:2500 dilutions in 5%Blotting Grade Blocker-TBS-T for 1 h at room temperature. β-Actin (abcam), Vinculin (Cell Signaling) or GAPDH (abcam) primary antibody for equal loading analysis was used in 1:1000 dilution in 5% Blotting Grade Blocker in TBS-T for 1 h at room temperature. For visualization of the results, chemiluminescence was performed with ECL. The membranes were visualized using LI-COR Fc. Sample bands were visualized with Chemi light exposed for 10 min and 700 light exposed for 1 min for the protein standards (ladder). All proteins were run in the same gel with their loading controls and only physical application was vertical cropping. All blots were evenly treated with brightness and contrast modifications (mostly + %20 Contrast and + %20 brightness of Office Powerpoint was applied.) and no bands were exaggerated or lost during the processing. To quantitatively analyze the band intensities, ImageJ was uses, statistical analysis were done with GraphPad PRISM.
Statistical analysis. Statistical analysis was performed with GraphPad PRISM encoded tests. Most of the data was analyzed with unpaired two-tailed t-test and also significancy of compound treated groups were validated with one-way ANNOVA test comparing to DMSO treatment groups. For (*) p < 0.05, (**) p < 0.01, (***) p < 0.001 and (****) p < 0.0001.

Data availability
All data generated or analyzed during this study are included in this published article.